There is a continuing need in medical practice, research and diagnostic procedures for rapid and accurate detection or quantification of biological and chemical substances which are present in biological fluids, cells or tissues. For example, the presence of drugs, hormones, steroids, polypeptides, nucleotides, prostaglandins, proteins, carbohydrates or infectious organisms (bacteria, fungi or viruses) in biological specimens has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
For example, organisms classified as gram positive bacteria, such as group specific Streptococcus, are known to be pathogenic in humans. For instance, Group A organisms are primarily responsible for causing B-hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac sequelae, glomerulonephritis, septic sore throat and puerpueral sepsis. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence at an early stage so that an appropriate course of treatment can be pursued. In most cases, the diagnostic tests require several hours, or at least up to 30 minutes. Even this limited wait may be intolerable in many instances where the practitioner has many waiting patients, and the patients themselves can not wait for the diagnosis without considerable cost, inconvenience or discomfort.
To provide diagnostic determinations, various methods have been devised for isolating and identifying biological or chemical substances employing specific binding reactions between the substance to be detected (identified as a "target ligand" or simply "ligand" herein) and receptors (molecules which specifically react or bind with that substance). This reaction between a ligand and its corresponding receptor is known as a specific binding reaction. Where either the ligand or receptor is an antibody, the reaction is known as an immunological reaction. More than one ligand or receptor may participate in each reaction.
Such reactions are detected in a number of ways. Generally, one or more participants of the specific binding reaction is detectably labeled. That is, it is either chosen because it is inherently detectable, or a detectable moiety (for example, an enzyme, radioisotope, chromogen or fluorogen) is incorporated therein in some manner. Many assays (for example, ELISA) today utilize enzymes as detectable moieties because of the convenience they provide in requiring minimum equipment and skills needed for the assays, as well as improved sensitivity in some cases.
Copending and commonly assigned U.S. Ser. No. 206,257, filed on even date herewith by Warren III and Snyder and entitled "Specific Binding Composition Comprising a Low pI Protein or Carbohydrate and a Diagnostic Test Kit and Method of Use" describes the use of a low pI protein with a specific binding species to reduce assay background without affecting sensitivity. This advance in the art greatly improves the assay. However, it has been found that further sensitivity is needed in addition to low background for the assay to effectively detect low amounts of ligand. This is especially critical where the ligand is a pathogen and early detection of disease is urgently needed.
Copending and commonly assigned U.S. Ser. No. 884,329 (filed July 10, 1986 by McClune) describes the use of a phenol or aniline in combination with peroxidase or a peroxidase-labeled ligand analog and a leuco dye. The phenol or aniline (such as 4'-hydroxyacetanilide) is used as a electron transfer agent to accelerate the formation of dye. At least 0.001 mmolar, and preferably 0.1 to 10 mmolar phenol or aniline is used in the composition. This application teaches that 4'-hydroxyacetanilide or other phenols will increase the rate of dye formation from leuco dyes so that the assay is more rapid. As more of the phenol is used, the more rapid the rate of dye formation. Moreover, the phenol and leuco dye are used in combination with a peroxidase-labeled ligand because of the nature of the assays described therein.
Generally, high concentrations of peroxidase-labeled antibody are needed to detect low concentrations of antigen in an assay. Higher concentrations are also conventionally used to achieve maximum complexing kinetics and to provide sufficient enzyme to maximize the overall kinetics of dye formation for a rapid and sensitive assay. Unfortunately, these higher concentrations of labeled antibody give higher background levels which can adversely affect sensitivity. Background is unwanted dye signal which is not the result of the presence of antigen.
Moreover, when the concentration of 4'-hydroxyacetanilide is increased as taught in U.S. Ser. No. 884,329 (noted above) to increase the rate of dye formation from a leuco dye (and make the assay more rapid), higher background levels also result.
Thus, it would be desirable to be able to increase the amount of enzyme-labeled antibody to detect low concentrations of antigen and to shorten assay time, but without adversely affecting sensitivity or increasing background.